Alpha-1 proteinase inhibitor (a1PI), also named alpha-1-antitrypsin (AAT), is a glycopeptide inhibitor of proteases, and is found in human serum and other fluids. Protease inhibition by a1PI is an essential component of the regulation of tissue proteolysis, and a1PI deficiency is implicated in the pathology of several diseases. Individuals who inherit an a1PI deficiency, for example, have increased risk of suffering from severe early-onset emphysema, the result of unregulated destruction of lung tissue by human leukocyte elastase. The administration of exogenous human a1PI has been shown to inhibit elastase and is associated with improved survival and reduction in the rate of decline of lung function in a1PI-deficient patients (Crystal et al., Ain. J. Respir. Crit. Care Med. 158: 49-59 (1998); R. Mahadeva and D. Lomas, Thorax 53: 501-505 (1998) for a review.)
Because of its therapeutic utility, commercial a1PI production has been the subject of considerable research. Much progress has been made in the production of recombinant AAT in E. coli (R. Bischoff et al., Biochemistry 30: 3464-3472 (1991)), yeast (K. Kwon et al., J. Biotechnology 42: 191-195 (1995); Bollen et al., U.S. Pat. No. 4,629,567), and plants (J. Huang et al., Biotechnol. Prog. 17: 126-33 (2001)), and by secretion in the milk of transgenic mammals (G. Wright et al., Biotechnology, 9: 830-834 (1991); A. L. Archibald, Proc. Natl. Acad. Sci. USA, 87: 5178-5182 (1990)).
However, isolation of a1PI from human plasma is presently the most efficient practical method of obtaining a1PI in quantity, and human plasma is the only FDA-approved source.
A number of processes for isolating and purifying a1PI from human plasma fractions have been described, involving combinations of precipitation, adsorption, extraction, and chromatographic steps.
Most published processes for a1PI isolation begin with one or more fractions of human plasma known as the Cohn fraction IV precipitates, e. g. Cohn fraction IV-1 or fraction IV1-4, which are obtained from plasma as a paste after a series of ethanol precipitations and pH adjustments (E. J. Cohn et al., J. Amer. Chem. Soc., 68: 459-475 (1946)).
M. H. Coan et al., Vox Sang., 48 (6): 333-342 (1985) describe a method in which a1PI was purified from frozen Cohn fraction IV-1 paste by polyethylene glycol precipitation and DEAE-Sepharose chromatography. R. H. Hein et al., Eur. Respir. J. Suppl., 9: 16s-20s (1990) also used Cohn IV-1 paste as starting material for commercial production of a1PI in a similar process as disclosed in Coan et al.
WO 95/35306 discloses a method starting from Cohn fraction IV1+IV4-precipitate obtained as described therein, which is subjected to a polyethylene glycol/ZnCl2-precipitation. Said precipitation is followed by another ZnCl2-precipitation before the re-solubilized precipitate is applied to a QAE-column.
WO 98/00154 teaches a process wherein a washing step has been included subsequently to Cohn-fractionation and before subjecting the a1PI-comprising material to further processing steps. In this method the IV1+IV4-paste is suspended in water or saline solution. Soluble proteins including albumin, globulins such as alpha-1-globulin and beta-globulin are then separated from the insoluble proteins comprising a1PI by filtration, centrifugation or the like. The residue is washed with water or saline solution to remove additional soluble protein physically trapped in the insoluble paste. Similar as described in WO 95/35306, the material is then subjected to PEG- and ZnCl2-precipitations and applied to an ion-exchange chromatography step.
As mentioned above, several other processes for a1PI from human plasma fractions have been described in the art.
However, while a1PI is an effective treatment for emphysema due to alpha-1-antitrypsin deficiency, treatment is very costly (currently about $25,000 per year), due to the limited supply and a complex manufacturing process. There remains a need for more efficient and cost-effective methods for isolating human a1PI from plasma. The present invention addresses this need.